Various scientific and scholarly articles are referenced throughout the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains.
The Ca2+ dependence of vertebrate skeletal muscle contraction is due entirely to a set of specialized accessory proteins closely associated with actin filaments. If myosin is mixed with pure actin filaments in a test tube, myosin ATPase is activated whether or not Ca2+ is present; in a normal myofibril, on the other hand, where the actin filaments are associated with accessory proteins, the activation of the myosin ATPase depends on Ca2+.
One of these accessory proteins is a rigid rod-shaped molecule, called tropomyosin because of similarities to myosin in its x-ray diffraction pattern. Like the myosin tail, tropomyosin is a dimer of two identical α-helical chains, which wind around each other in a coiled coil. By binding along the length of an actin filament, tropomyosin stabilizes and stiffens the filament.
Tropomyosins are present in all eukaryotic cells. Different isoforms of tropomyosin, generated through alternative splicing, are expressed in a tissue-specific manner (Less-Miller, J P, et al., Bioassays 1991; 13: 429–37). In human fibroblast tissue, at least eight isoforms of TMs have been identified. These isoforms range in molecular weights from 30–40 kDa (Lin J J-C, et al., Int Rev Cytol 1997; 170:1–38). Classically, tropomyosins are known to remain intracellular because they lack the signal sequence required for membrane insertion and translocation (Less-Miller, supra).
Human tropomyosin (hTM) is a cytoskeletal microfilament protein. A significant number of ulcerative colitis patients show a preferential immune response to hTMs, in particular, the hTM5 isoform. Thus, hTM is a candidate autoantigen in ulcerative colitis. Using lamina propria lymphocytes from mucosa of patients with ulcerative colitis and ulcerative colitis sera, an autoantibody response to hTM isoforms has been demonstrated in several independent studies, including that of Das, K M, et al. J Immunol. 1993; 150:2487–93. Such an anti-hTM autoantibody response, however, was not seen in patients with Crohn's disease. Recently, these findings were extended to an animal model of colitis using TCRα−/− mice (Mizoguchi, A., et al. J. Exp Med. 1996; 183: 847–56). Severity of colitis in these mice is directly correlated with the increased titer of anti-TM autoantibodies and the increased number of appendicular B cells producing anti-TM autoantibodies (Mizoguchi, A., et al. J. Exp Med 1996; 184:707–15).
In colon epithelium, the most predominantly expressed hTM isoform is hTM5 (Geng X, et al., Gastroenterology 1998; 114:912–22). It is presently unknown whether hTM5 is accessible to anti-TM autoantibodies, particularly when the target protein is expected to be exclusively intracellular. The possibility of externalization of hTM5 in colon epithelium and likelihood of the passive transport of hTM5 with a secretory protein has been considered. One likely candidate for this chaperone function is a colon epithelial-specific protein recognized by the 7E12H12 monoclonal antibody.
The monoclonal antibody 7E12H12 was raised using highly enriched colonic tropomyosin (earlier named as 40 kDa protein or p40) (Das K M, et al., J. Immunol 1987; 139:77–84). However, 7E12H12 does not react with any of the known hTM isoforms in ELISA or immunotransblot analysis, either from muscle as well as from non-muscle epithelial cells (Das K M, et al., Gastroenterology 1997; 112:A955). However, the 7E12H12 monoclonal antibody recognizes a cell membrane associated protein present exclusively in the colon epithelium (Das K M, et al. (1987) supra; Das K M, et al. (1997) supra). By immunotransblot analyses, CEP has been identified as a high molecular weight (>200 kDa) protein present in colon epithelial cells but not in small intestinal enterocytes. Among the colon cancer cell lines, LS-180, and DLD-1 cells express the 7E12H12-reactive protein but HT-29 cells do not (Hassan T., et al., Clin Exp Immunol. 1995; 100:457–62).